The superfamily of "regulator of G-protein signaling" (RGS) proteins share a defining RGS domain that accelerates the intrinsic GTP hydrolysis rate of heterotrimeric G-protein alpha subunits - an enzymatic activity that terminates signaling by activated G-protein coupled receptors (GPCRs). As GPCRs constitute the largest single class of protein target for existing drugs, small molecule modulators of RGS protein action should hold great promise for the development of novel therapeutics, yet no bona fide proof-of-principle small molecule has yet been developed. We recently created a novel, robust, and facile method of measuring RGS domain GTPase-accelerating function using fluorescence polarization. In lieu of the traditional, cumbersome, radioactive "single-turnover" assay, we have developed a high- throughput screening (HTS) method based on detecting RGS-accelerated GDP production by a G subunit with altered nucleotide binding and hydrolysis rates. This enzymatic assay differs considerably from the RGS/G protein-protein interaction assays that have previously been screened by the MLPCN. We request funding to deliver this HTS assay (including required protein reagents) to an MLPCN, as well as to facilitate our lab's post-screening analysis of hits using various medium-throughput secondary and counterscreen assays. PUBLIC HEALTH RELEVANCE: A particular class of cell-surface proteins, the G protein-coupled receptor superfamily, has for many decades provided valuable targets for drug discovery across a variety of clinical needs and diseases. A large family of negative regulators of these receptors (the "RGS proteins") was discovered over ten years ago, but their potential as additional drug discovery targets has yet to be tested owing to the present dearth of chemical modulators of their action. Here we describe a novel, enzymatic reaction for the high-throughput screening of small molecule libraries for modulators of RGS protein action;our intent is to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) with the assistance of one of the nodes within the NIH Molecular Libraries Probe Production Centers Network (MLPCN). Our long-term goal is to develop novel chemical probes and (ultimately) drugs that act at the level of the RGS protein to improve therapy of multiple pathological conditions caused by aberrant GPCR signal transduction.